Extracellular matrix bioink boosts stemness and facilitates transplantation of intestinal organoids as a biosafe Matrigel alternative

Abstract Organoids hold inestimable therapeutic potential in regenerative medicine and are increasingly serving as an in vitro research platform. Still, their expanding applications are critically restricted by the canonical culture matrix and system. Synthesis of a suitable bioink of bioactivity, biosecurity, tunable stiffness, and printability to replace conventional matrices and fabricate customized culture systems remains challenging. Here, we envisaged a novel bioink formulation based on decellularized extracellular matrix (dECM) from porcine small intestinal submucosa for organoids bioprinting, which provides intestinal stem cells (ISCs) with niche‐specific ECM content and biomimetic microstructure. Intestinal organoids cultured in the fabricated bioink exhibited robust generation as well as a distinct differentiation pattern and transcriptomic signature. This bioink established a new co‐culture system able to study interaction between epithelial homeostasis and submucosal cells and promote organoids maturation after transplantation into the mesentery of immune‐deficient NODSCID‐gamma (NSG) mice. In summary, the development of such photo‐responsive bioink has the potential to replace tumor‐derived Matrigel and facilitate the application of organoids in translational medicine and disease modeling.


| INTRODUCTION
The intestinal epithelium hosts short-lived differentiated cells of diverse lineages with a renewal cycle of 4-5 days and immortalized proliferative ISCs, which reside at the bottom of crypts. 1 This subtle homeostasis of the epithelium depends on bidirectional gradients of proliferative and differentiated signals established by interspersed Paneth cells and intestinal stromal components, including intestinal subepithelial myofibroblast (ISEMFs), macrophages and endothelial cells, which are involved in the maintenance of ISCs via intracellular pathways such as Wnt/R-spondin, BMP and Notch signaling. [2][3][4][5] The identification of leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) as a marker of ISCs enabled isolation and in vitro expansion of ISCs. 6 Later, Sato et al. 7 found that ISCs exhibited a regular expansion and differentiation pattern and formed self-assembly 3D aggregates within Matrigel, an extract from Engelbreth-Holm-Swarm sarcoma with low mechanical properties (354.50 ± 29.37 Pa) ( Figure 1e). 8 Such ISC-based micron-sized 3D multicellular constructs with projecting crypt-like buds and sealed-off lumen are named intestinal organoids.
Despite the potentials in regenerative medicine and disease modeling, conventional organoid culture patterns have limitations. 9,10 First, encapsuled organoids within the hydrogel dome lack maneuverability and homogeneity. Second, canonical organoid culture pattern hardly reaches designed deposition of ISCs and refined tissue models for therapeutic application. 11 Third, spatially variant biochemical distribution caused by dome-like constructs can result in different maturation and differentiation levels of cultured organoids. 12 Some of these drawbacks are attributed to Matrigel. 13 As an exclusive ECM analog for organoid culture, Matrigel has increasingly imposed constraints on organoid applications. Potential contamination resulting from tumor origin leads to uncertain biosecurity and tumorigenicity.
Component variations among batches cause low homogeneity and reproducibility. What is more, the entactin-mediated gelation of Matrigel is not amenable to chemical modification to regulate mechanical properties, which is not applicable for reprocessing such as bioprinting. 14 There is an urgency to synthesize alternatives and fabricate new culture matrices with tunable biophysical properties to expand organoid research and application. 15 Two major solutions exist when it comes to Matrigel alternatives. 16 Gjorevski et al. 17 combined a well-defined 3D matrix based on multiarmed-polyethylene glycol (PEG) macromers and peptides from fibronectin to fully recapitulate key cues dominating ISC expansion.
The synthetic hydrogel with customized contents and tunable mechanical properties (300-1000 Pa) exhibits moderate organoids generation efficiency. 18,19 Roh et al. 20 selected natural silk protein to bioengineer epithelial scaffolds. Other natural biomaterials such as hyaluronic acid (HA) and collagen gels also hold potential in organoid culture. 21,22 Here, we envisaged and fabricated a new bioderived hydrogel with enhanced printability consisting of (i) decellularized extracellular matrix (dECM) pregel from porcine small intestine; (ii) photoresponsive gelatin methacrylate (GelMA); (iii) photo-initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP); and (iv) thickener HA. dECM is a bioactive scaffold from native tissues with cells, functional enzymes and partial biochemical factors removed. 23 After proper decellularization processes, dECM material consisting of collagens, elastin, fibronectin, and laminin could offer a biomimetic environment with retained native microstructure, cell-ECM interactions, and minor immunogenicity. As a US Food and Drug Administration-approved medicinal product in use, dECM provides a potential transplantation vector of organoids for regenerative medicine. To enhance the printability of dECM hydrogel, we replenished a biosafe GelMA-based crosslinked network. 24,25 GelMA inks are widely utilized in 3D printing for their rapid gelation kinetics and good photo-curability. Compared to other photopolymers such as PEG diacrylate, poly-(acrylic acid), or elastic resins, GelMA contains inherent Arg-Gly-Asp (RGD) sequences, which are required for organoid encapsulation and proliferation. Also, HA is added to increase the biocompatibility and viscoelasticity and facilitate the cell proliferation and migration. 26 To verify this hypothesis, we fabricated dECM powder and assessed decellularization effectiveness. Afterward, we prepared dECM-based bioinks with varied GelMA concentrations and rheological characteristics. We isolated primary small intestinal crypts from C57BL/6 mice and compared in vitro organoid formation in the bioink with that in Matrigel. Differentiation pattern and transcriptomic signature between two groups were analyzed accordingly. A feasible co-culture system was established via bioprinting of selected bioinks to investigate the crosstalk between printed intestinal organoids and submucosal cells. Furthermore, dECM-based bioinks containing organoids were transplanted into the mesentery of immune-deficient NSG mice to verify its applicability for regenerative therapy (Scheme 1).

| Decellularization and characterization of dECM material
As a tissue-specific material, components of dECM vary according to animal species and gender, tissue origin, and decellularization strategy. 27 The small intestine tissue from Landrace piglets was used in this study. In order to preserve protein structure under the premise of thorough decellularization, a conjoint processing strategy based on previous published protocols was modified consisting of (i) tissue harvest and rinse to remove mesentery, external layer, and mucosa; (ii) sodium deoxycholate (SDC) and deoxyribonuclease I (DNase-I) treatment to remove cells and residual DNA; (iii) lyophilization and milling into fine powder; (iv) digestion in pepsin and HCl to gain pregel; and (v) change of pH, salinity, and temperature to initiate gelation ( Figure 1a). 24 A qualitative analysis of decellularized tissue by histological section staining was carried out. Staining of hematoxylin and eosin (H&E) validated the removal of cell components such as nuclei. Staining of Alcian Picrosirius Red (PR) and blue-periodic acid-Schiff (AB-PAS) confirmed the preservation of glycosaminoglycans (GAGs) and multitype collagens ( Figure 1b). To meet the standard of dECM, a quantitative analysis of residual DNA in tissue was carried out after SDC and DNase-I treatment, which showed a significant decrease (Figure 1c). Final DNA content was less than the permitted maximum value 50 ng/mg. 28 To identify major constituents of dECM powder, a quantitative analysis of collagen, laminin, and elastin was conducted by ELISA. Collagen and elastin showed increased mass proportions compared with those in fresh tissues ( Figure 1d). dECM was digested as 6, 8, 10, and 12 mg/ml, whose compressive modulus and turbidimetric gelation kinetics were analyzed.
Next, we tested the culture efficacy of mouse intestinal organoids within dECM gels. Isolated small intestinal crypts from C57BL/6 mouse were dispersed in the pregels of dECM. Notably, in the first passage, 10 mg/ml dECM gel showed comparable culture efficacy to Matrigel, while the others were inferior to Matrigel (Figure 1g). Among dECM gels, 10 and 12 mg/ml dECM gels could better support the formation of organoids than 6 mg/ml dECM gel.

| Fabrication and characterization of dECMbased bioinks
The mechanical properties, shear-thinning behavior, and viscosity of a bioink are strongly associated with its printability and precision of the construct. 29 To synthesize an ECM-based bioink with suitable bioactivity and printability, we chose LAP-triggered chemical crosslinking between methacrylates, HA, and glycerol to improve the mechanical properties of 10 mg/ml dECM gel, which showed comparable culture efficacy to Matrigel (Figure 2a). We investigated the microstructure of dECM-inks after gelation. Representative SEM images showed porous microstructures ( Figure 2b). 30 Oscillatory rheological characteristics of dECM-inks were assessed. dECM-inks exhibited highly tunable mechanical properties, whose storage modulus (G') and loss modulus (G") increased with GelMA concentration in frequency-sweep ( Figure 2c). What is more, the adjunction of HA could improve the mechanical strength of dECM-inks. In time-sweep, dECM-inks all underwent a swift gelation (~5 s), which indicated a rapid responsiveness to blue light (25 mW/cm 2 ).
In addition, all dECM-inks exhibited a modest shear-thinning behavior and HA could increase the viscosity (Figure 2f), which meant that dECM-HA-inks were more suitable for extrusion-based 3D printing. Elastic modulus or stiffness of ECM largely influences or regulates fundamental cellular processes, such as growth, proliferation, migration, differentiation, and organoid formation, making elastic modulus a major index in biomaterial assessment. In this study, elastic modulus of each fabricated bioink was measured through compressive testing ( Figure 2g). dECM-inks exhibited varied but tunable modulus (modulus of dECM-ink I: 240. 23  A 2D patterning test was performed by printing a lattice pattern with 800 Â 800 μm 2 pores, which is one of the most used constructs in tissue engineering. dECM-ink I and dECM-HA-ink I produced unstable constructs ( Figure 2h). However, dECM-HA-ink II and III showed better printability and shape fidelity compared to their parallels (Figure 2i).

| dECM-based bioinks enable the formation of mouse intestinal organoid
We then explored mouse intestinal organoids culture efficacy of fabricated ECM-based bioinks (Figure 3a). Isolated small intestinal crypts from C57BL/6 mouse were dispersed in pregels of dECM-HA-ink I, II, S C H E M E 1 Schematic diagrams of decellularization and fabrication processes of decellularized extracellular matrix (dECM)-based bioink and blue light-induced bioprinting method and III, which showed better printability and higher viscosity than their parallels. A canonical factor cluster consisting of R-spondin, epidermal growth factor (EGF) and Noggin was used as described previously. 7 ISCs from mouse small intestine showed favorable adaption to dECM-HA-inks, which had similar formation organoids counts at Day 7 of two consecutive passages to that of Matrigel. Along with cell passage, the number of formed organoids exhibited a decrease. In Passage 3, dECM-HA-ink II showed better culture efficacy compared to dECM-HA-ink I and III, indicating a higher proliferation level. For morphological assessment, diameters of organoids at Day 7 of three consecutive passages were analyzed (Figure 3b). In Passage 3, organoids within dECM-HA-ink II were characterized by significantly larger size, which also indicated a higher proliferation level. 31 Budding and expansion of formed organoids at different times of culture were observed by bright field photography (Figure 3c). In all formed organoids, enterocysts accounted for a significantly higher F I G U R E 1 Characterization of decellularized extracellular matrix (dECM) hydrogel. (a) The preparation and gelation processes of dECM powder including harvesting of small intestine from freshly killed piglets, decellularization of the submucosa, lyophilization and milling into powder, sterilization, digestion in pepsin and HCl, adjustment of pH and salinity and incubation at 37 C. (b) Qualitative analysis of decellularized small intestine tissue by histological section including hematoxylin and eosin (H&E) for nucleus, Picrosirius Red (PR) for collagens and Alcian blueperiodic acid-Schiff (AB-PAS) for glycosaminoglycans (GAGs). Scale bar 100 μm. (c) Quantitative analysis of DNA content in fresh untreated intestinal tissue, sodium deoxycholate (SDC)-treated submucosa, and DNase-treated submucosa. Mean ± SD (n = 3 batches). One-way ANOVA. *p < 0.05 and ****p < 0.0001. (d) Quantitative ELISA analysis of dECM pregel including collagen, laminin, and elastin. Mean ± SD (n = 3 batches). Two-sided t-test *p < 0.05. (e) Compressive modulus measured by compressive testing. Mean ± S.D. (n = 3 samples). One-way ANOVA. *p < 0.05 and **p < 0.01. (f) Turbidity analysis of dECM gels and Matrigel by spectrophotometry during heat-mediated gelation. (g) Formed intestinal organoids per field of view at 100Â at Day 7 at first passage. Mean ± S.D. (n = 16 from four organoids cultures). One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (h) Typical bright field images of formed organoids at Day 5 at first passage within dECM hydrogels. Scale bar 100 μm Wnt/R-spondin signaling in the early stages was considered to cause enterocyst formation. 33 However, dECM-HA-ink I, II, and III exhibited a significantly lower ratio of enterocysts, which indicated that dECM hydrogel could deliver biochemical stimulus and maintain stemness of ISCs more effectively and uniformly compared with Matrigel. 12

| Organoids within dECM-based bioinks exhibit distinct differentiation pattern
To further characterize cultured intestinal organoids and ISC differentiation in dECM-HA-ink II, which showed higher culture efficacy than dECM-HA-ink I and III, we selected representative intestinal epithelial markers and observed their expression and location by immunofluorescence ( Figure 4a). ISCs in dECM-HA-ink II were capable of differentiating into enterocytes, goblet cells, enteroendocrine cells, and Paneth cells.

| Transcriptomic analysis of organoids within dECM-based bioink and Matrigel
To further reveal the cellular behavior and differentiation feature in dECM-based bioink, we performed RNA sequencing on formed intestinal organoids ( Figure S1a). 31,35 In principal component analysis were significantly upregulated in organoids of dECM-HA-ink II. Some common crypts markers (e.g., WNT7A and LYZ2) were upregulated in dECM-HA-ink II group, which indicated enhanced ISCs function.
Formation of organoids at Day 7 in the +ISEMFs group was significantly higher than that in the Ctrl group (Figure 6c). However, organoid formation was significantly decreased in the +M1 group after Day 1 (Figure 6d). To reveal cell expansion level, live/dead staining was performed. In immunofluorescence photographs with printed lines at the center, living cell aggregates were seen in the +ISEMFs, + MØ and Ctrl groups (Figure 6e). However, most cells were dead in the +M1 group, which indicated that epithelial cells were sensitive to inflammatory effects induced by M1 macrophages. 37 +ISEMFs and + MØ groups were characterized with significantly fewer dead cells (Figure 6f).
To reveal ISC proliferation and differentiation level, qPCR was carried out focusing on representative intestinal markers (Figure 6g). For

ISC markers
LGR5 and OLFM4, their mRNA expression was upregulated compared to that in the Ctrl group, which confirmed enhanced ISC stemness with ISEMFs and MØ macrophages. It has been reported that subepithelial myofibroblasts and macrophages activate Wnt/Rspondin signaling as a Wnt source to maintain epithelial homeostasis. 4 However, differentiation markers such as KRT20, VIL1, FABP2, CHGA and MUC2 were increased in the +MØ group compared to +ISEMFs group, which indicated a differentiation-promoting effect of residual macrophages in submucosa. What's more, in the +M1 Next, we performed another in vivo delivery experiment with organoids seeded in the pregels of Matrigel and dECM-HA-ink II. 39 We chose 4-week-old male NSG mice for organoids transplantation to avoid reject reaction toward allogeneic cells and observe long-term survival. And we selected mesentery which served as a physiological and anatomic engraftment site (Figure 8a). 39 After transplantation, the grafts were harvested at Days 3, 5, 7, and 14, respectively, to measure their change in volume, which was relevant to the maturation of organoids within. Notably, dECM grafts suffered a volume loss significantly less than the Matrigel group which might be caused by higher

| Preparation of porcine small intestinal submucosal tissue
Fresh whole small intestine of male Landrace (Sus scrofa domesticus) piglets up to 3 kg was purchased from Jiangsu Hurun Agricultural Products Co. Ltd. Decellularization of the whole small intestine submucosa was performed according to reported protocols with some modifications. 43 The mesentery and external layer of the small intestine were removed. The internal layer consisting of mucosa and submucosa was cut off longitudinally and fully rinsed and cleaned with pressurized water. Later, a scalpel handle was used to scrape off fluffy mucosa. Submucosal tissue was cut into 5-cm pieces.

| Fabrication of dECM from porcine small intestinal submucosa
Each batch of dECM was fabricated from three piglets' submucosal tissue. A conjoint decellularization strategy modified from established protocols for porcine intestine was used subsequently. To initiate decellularization, submucosal tissue pieces were placed in pure water Biosharp, Anhui, China). Acquired dECM powder was stored at À20 C until further use.

| Tissue histology
Samples were taken randomly from decellularized submucosa to initiate paraffin embedding. Samples were fixed in 4% paraformaldehyde solution in PBS for 1 h at room temperature, dehydrated, paraffin embedded, and cut into 5-μm sections. H&E staining was performed on tissue slides to confirm the absence of nuclei. AB-PAS and PR were used to assess the presence of GAGs and collagen, respectively.

| Quantification analysis of DNA
Samples were taken at random after harvesting, after SDC treatment and after DNase-I treatment. DNA content was assessed by a Pur-eLink Genomic DNA Mini Kit (K182000; Thermo Fisher). Final DNA concentration was measured by a NanoDrop microvolume spectrophotometer (ND-ONEC-W; Thermo Fisher). Samples from three different batches were tested.

| Turbidity
Pregel samples from dECM-gel and Matrigel (356231; Corning) was taken to measure turbidity using a spectrophotometer (PT-3502PC; Potenov). Two hundred microliters of pregels were pipetted into a 96-well plate. Absorbance at 450 nm was measured at 37 C once per min for 1 h. Measured results were standardized to a PBS control group to calculate normalized absorbance (NA) using the formulation below. R is the absorbance reading measured at a selected time. R min is the smallest absorbance reading. R max is the highest absorbance reading.
NA ¼ R À R min R max À R min

| SEM analysis
Hydrogel samples were prepared (4 mm in diameter and 10 mm in height). The samples were frozen at À80 C and then freeze-dried during 48 h. The specimens were cracked, sputter-coated with Pt and examined using SEM (S-3400N; Hitachi, Tokyo, Japan).

| Real-time qPCR
After total RNA harvest, cDNA was prepared using Superscript IV cDNA synthesis kit (Thermo Fisher). 32 Real-time PCR was performed using a QuantStudio 6 Flex (Applied Biosystems, Foster City, CA, USA). Primer sequences are listed below.

| Culture of ISEMFs and macrophages
ISEMFs were isolated based on published protocols. 5 After euthanasia via cervical dislocation, rinsed small intestinal tissue from 7 day-old male C57BL/6 mice was cut into 0.5-mm 2 pieces and transferred into a T25 flask. Dispase at 0.31 mg/ml (17105041; Gibco, Grand Island, NY, USA) was used to digest rinsed tissue. After incubation at room temperature for 30 min on rotation, the supernatant underwent multistep centrifugation to acquire pellets containing ISEMFs. Medium for ISEMFs consisted of DMEM high-glucose (KGA12800; KeyGEN BioTECH), 15% fetal bovine serum, 1% penicillin/streptomycin and EGF 50 ng/ml.
To acquire BMDMs, BM was extracted from the tibia and femur of 4-week-old male C57BL/6 mice following removal of surrounding muscle. 46 The bone marrow was flushed into a Petri dish using a 1-ml syringe filled with PBS. Cell suspension was centrifuged (250 Â g) at room temperature for 5 min to pellet cells. density of 1 Â 10 6 /ml, respectively. 20 The submucosal cell-laden pregel was transferred into a glass bottom dish. The crypt-laden dECM-HA-ink II was extruded in the layer of dECM-free pregel. After printing, blue light was used to initiate gelation of two hydrogels within the dish. No submucosal cells were added to the Ctrl group.
For the +MØ group and + M1 groups, we used organoid culture medium supplemented with 50 ng/ml M-CSF.

| In vivo transplantation
Four-week-old male NSG mice (n = 24) were anesthetized using 4% chloral hydrate via intraperitoneal injection. An abdominal incision (1.2 cm) was performed to find ileocecal valve and expose nearby mesenteries. For dECM-HA-ink II group (n = 12), 4000 matured organoids which had been cultured for 3 days were seeded within 50 μl pregel of dECM-HA-ink II. Then the 50 μl dECM-HA-ink II pregel containing organoids was injected into the thickened part of the mesentery. The injection was followed by 10 s long exposure to blue light irradiation. For Matrigel group (n = 12), 4000 matured organoids, which had been cultured for 3 days were seeded within 50 μl pregel of Matrigel. And the mixture was injected into the thickened part of the mesentery. For both dECM-HA-ink II and Matrigel group, at Days 3, 5, 7 and 14 after transplantation, three mice were sacrificed via cervical dislocation for graft size measurement. Grafts harvested at Day 14 were quick-frozen in liquid nitrogen followed by immunostaining analysis. For organoids maturation demonstration, formed organoids on frozen sections from different grafts were counted (n = 3). Organoid diameters were also collected from different sections (n = 10).

| Statistical analysis
Differences between the experimental groups were analyzed using Two-sided t-test or one-way ANOVA. RNA sequencing relevant PCA analyze, pie plot and hierarchical clustering were performed on ANNOROAD (v. 2018). Genomic data were filtered using Microsoft Excel (v. 2016). Other graphs were performed on GraphPad Prism (v. 8.0).

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.